Loop-Mediated Isothermal Amplification as a Promising Method for Mass COVID-19 Diagnostics.

Real-time reverse-transcription polymerase chain reaction (RT-PCR) is currently the most popular method for early COVID-19 diagnostics. However, loop-mediated isothermal amplification (LAMP) is superior to real-time RT-PCR in rapidity and simplicity, since it does not require expensive laboratory equipment and trained personnel. LAMP-based diagnostic kits for COVID-19 testing already exist, but corresponding tests are not yet widely available. The method has great potential for mass application. Here, we discuss the technical and methodological aspects of its widespread adoption.

Detection of Low-Abundant MicroRNAs with Hybridization Microchips.

The effect of low concentrations of miRNA on the ability of GeneChip miRNA 4.0 hybridization chips to evaluate their representation in the sample was studied. It is shown that the evaluation of the expression of 61 miRNAs is statistically significantly associated with the multiplicity of plasma dilution. Only 12 miRNAs showed very high Pearson correlation coefficient (>0.95) and they all decreased in response to dilution. High abundance of has-miR-4532 miRNA in plasma was demonstrated. This miRNA was never detected during sequencing of similar samples. It was concluded that in case of miRNA expression <1.12±0.33 units in log2 scale, dilution was not followed by further decrease in the signal intensity in GeneChip miRNA 4.0 chips.

Changes in the Metastatic Properties of MDA-MB-231 Cells after IGFBP6 Gene Knockdown Is Associated with Increased Expression of miRNA Genes Controlling INSR, IGF1R, and CCND1 Genes.

Metastatic cascade is associated with the process of epithelial-mesenchymal transition accompanied by changes in cell proliferation, migration, adhesion, and invasiveness mediated by the insulin-like growth factor (IGF) signal pathway. IGFBP6 protein binds IGF and prevents its interaction with receptors. IGFBP6 gene knockdown through RNA-interference inhibits cell migration and increased the rate of proliferation of breast cancer MDA-MB-231 cells. IGFBP6 knockdown cells are characterized by increased expression of MIR100 and MIRLET7A2 genes encoding hsa-miR-100-3p, hsa-miR-100-5p, hsa-let-7a-5p, and hsa-let-7a-2-3p miRNA. The target genes of these microRNAs are IGF2, IGF1R, INSR, and CCND1 associated with IGF signaling pathway and proliferative and migratory activity during the metastatic cascade. A significant decrease in the expression of INSR and CCND1 genes was demonstrated by PCR and microarray analysis.

α-Fetoprotein Influence on the Conversion of Naïve T-Helpers into Memory T-Cell Effector Subpopulations.

The effect of native α-fetoprotein (AFP) on the conversion of naïve T-helpers into central memory T-cells (TCM) and effector subpopulations of the preterminally differentiated (TEM) and terminally differentiated (TEMRA) memory T-cells was studied. AFP was found to prevent the conversion of naïve T-helpers into effector subpopulations of memory T cells (TEM and TEMRA) while reducing the total production of IL-4 and IFN-γ by the studied cell populations. The data reveal a new role of AFP in the immune tolerance formation during pregnancy.

New Fluorescent Reporter Systems for Evaluation of the Expression of E- and N-Cadherins.

During metastatic growth, cells of solid tumors undergo phenotypical changes related to epithelial-mesenchymal transition. Epithelial-mesenchymal transition is regarded as a potential target for prospective antitumor drugs. Fluorescent reporter systems for evaluation of the expression of markers of epithelial and mesenchymal status (E- and N-cadherins) were created. The described approaches can be used for creation of analogous reporter systems.

Role of IGFBP6 Protein in the Regulation of Epithelial-Mesenchymal Transition Genes.

Protein IGFBP6 plays an important role in the pathogenesis of many malignant tumors, including breast cancer. The relationship between IGFBP6 protein and the expression of genes associated with the epithelial-mesenchymal transition is studied. Gene IGFBP6 knockdown does not trigger the epithelial-mesenchymal transition in MDA-MB-231 cells, but modifies significantly the expression of many genes involved in this process. A decrease of IGFBP6 expression can involve a decrease in the expression of N-cadherin and transcription factor Slug.

In Vitro Model for Studying of the Role of IGFBP6 Gene in Breast Cancer Metastasizing.

IGFBP6 gene plays an important role in the pathogenesis of breast cancer. In this work, we performed knockdown of IGFBP6 gene in MDA-MB-231 cells and obtained a stable cell line. Knockdown of IGFBP6 gene was confirmed by the real-time PCR. The influence of IGFBP6 gene on migration and proliferation of breast cancer cells was studied. Knockdown of IGFBP6 gene reduced migration activity of MDA-MB-231 cells and increased their proliferation rate. This in vitro cell model can be used for the further analysis of the role of IGFBP6 gene in the pathogenesis of breast cancer.

hsa-miR-1973 MicroRNA is Significantly and Differentially Expressed in MDA-MB-231 Cells of Breast Adenocarcinoma and Xenografts Derived from the Tumor.

The gene expression profiles of MDA-MB-231 cell line of breast cancer and xenografts derived from this tumor in murine model were analyzed using GeneChip Human Transcriptome array 2.0 platform (Affymetrix). A more than 1000-fold increase in the MIR1973 gene expression was observed in the xenografts compared to the original cell line. Real-time reverse transcription PCR showed that the content of mature hsa-miR-1973 microRNA encoded by this gene was elevated in the xenografts by more than 300 times. According to microarray analysis, none of hsa-miR-1973 target genes available in the databases changed the expression in the cell line and xenografts. A possible role of hsa-miR-1973 is reduction of apoptosis in the tumor.

Plasma Level of hsa-miR-619-5p microRNA Is Associated with Prostatic Cancer Dissemination beyond the Capsule.

Profiles of circulating microRNA in the plasma of patients with prostate cancer with pathomorphological stages pT2, pT3, and pT4 are analyzed. The level of circulating microRNA hsa-miR-619-5p is elevated in patients with extracapsular spreading of the tumor, increasing significantly from stage pT2 to stage pT4.

Detection of Potential Metastatic Prostate Cancer Circulating Biomarkers by Comparison of miRNA Profiles in DU145 Cells and Culture Medium.

We studied the profile of miRNA secreted into culture medium by DU145 prostate cancer cells and identified a subset of miRNAs characterized by the absence of correlation of their content in the cell and medium, which is likely a result of specific secretion. Three of these miRNA, hsa-miR-4417, hsa-miR-3175, and hsa-miR-6782-5p, exhibit the highest expression and are candidate circulating biomarkers for metastatic activity of prostate cancer. Two of these miRNA are coded by introns of genes linked with genome stability maintenance and chromatin remodeling regulation.

Maintenance of High Cytochrome P450 Expression in HepaRG Cell Spheroids in DMSO-Free Medium.

We studied the effects of DMSO and fibroblasts during HepaRG cell spheroid formation and conditions of their subsequent culturing on the levels of mRNA of the major cytochromes P450. A protocol of spheroid formation from differentiated HepaRG cells and their culturing in serum- and DMSO-free medium is developed.

MicroRNA hsa-miR-4674 in Hemolysis-Free Blood Plasma Is Associated with Distant Metastases of Prostatic Cancer.

We analyzed microRNA profile in hemolysis-free blood plasma of patients with prostatic cancer. The metastatic form of prostatic cancer was found to be associated with increased levels of hsa-miR-22-3p, hsa-miR-663a, and hsa-miR-4674 in comparison with non-metastatic form. Common candidate target genes of these microRNA include JUNB, KMT2A, and XPO6.

Plasma Levels of hsa-miR-619-5p and hsa-miR-1184 Differ in Prostatic Benign Hyperplasia and Cancer.

Peripheral blood plasma profiles of circulating microRNA expression were analyzed in patients with prostatic cancer and benign hyperplasia. In prostatic cancer, significant increase in hsa-miR-619-5p and hsa-miR-1184 microRNA expression and significant decrease in hsalet-7b-5p and hsa-let-7c-5p microRNA expression were observed. The role of the relationship between the microRNA expression and the activities and functions of host genes with introns encoding these microRNA is discussed.

Analysis of Plasma microRNA Associated with Hemolysis.

We analyzed the effect of hemolysis on microRNA profi le of blood plasma. It was found that hemolysis of ~0.05% erythrocytes in a sample signifi cantly affected the concentration of 9 microRNA: hsa-miR-486-5p, hsa-miR-16-5p, hsa-miR-451a, hsa-miR-106a-5p, hsa-miR-17-5p, hsa-miR-93-5p, hsa-miR-20a-5p, hsa-miR-107, and hsa-miR-20b-5p. The effect of hemolysis on plasma content of miR-17 family microRNA was demonstrated.

Localization and Expression of Nucleoside Transporters ENT1 and ENT2 in Polar Cells of Intestinal Epithelium.

Сасо-2 cell line is widely used in in vitro studies of the intestinal wall permeability for drugs, but transport activity of these cells has not been thoroughly studied. We analyzed localization and expression of nucleoside transporters of the ENT family transferring a number of anticancer and antiviral drugs. Immunocytochemical staining revealed apical localization of ENT1 and integral expression of ENT2 in Caco-2 cells. Based on these data and on the value of hypoxanthine uptake by these cells, we created a mathematical model allowing quantification of transporter expression on the apical and basolateral membranes of Caco-2 cells. The discrepancy between gene and protein expression of the transporter complicating prediction of patient's sensitivity to the drug upon individual therapy selection is discussed.

Profile of microRNA in Blood Plasma of Healthy Humans.

Analysis of the plasma microRNA profile can be used for the diagnosis of various pathological and physiological conditions. Complete microRNA microprofiling is an extremely important task. Here we used microarray analysis allowing measurement of the expression of 2500 microRNA (MirBase, version 20). About 10% known microRNA were found in the plasma. Most of the detected microRNA (69 microRNA; ~30%) were encoded by mirtrons.

Effects of cadmium chloride on the functional state of human intestinal cells.

Toxic effects of cadmium chloride in concentration range from 1 to 300 µM on differentiated human intestinal epithelial Caco-2 cells after three hours of exposure were investigated. Processes of disorganization of the actin cytoskeleton associated with the toxic effects of cadmium were characterized by fluorescent microscopy. The cadmium-induced activation of cellular stress response processes (changes in the mRNA expression of caspase-3, heat-shock and oxidative stress genes) has been demonstrated. The study revealed dose-dependent changes in mRNA expression levels of proteins involved in the formation of adherens (E-Cadherin and p120 catenin) and tight intercellular junction contacts (Claudin 4 and ZO1). The time- and concentration-dependent trend of cell monolayer transepithelial resistance lowering, characterizing the loss of intercellular contacts density with prolongation of cell exposure cadmium chloride was estimated. Results indicates that proteins associated with tight and adhesion junctions are primary targets of cadmium. Amongst genes involved in cell junction formation, the genes encoding E-Cadherin and p120-catenin proved to be the most sensitive to cadmium influence.

Expression of stress-dependent genes in hepatocytes spheroids after cisplatin treatment.

Effects of cisplatin on the hepatic HepaRG cells cultured in spheroids were estimated using biochemical, cytofluorometric, and molecular methods. Hepatocyte viability and expression of mRNA of stress-dependent genes involved in the cell response to toxic agent cisplatin underwent time- and dose-dependent changes. Activation of oxidative stress was observed at the early stages of incubation (3 h) followed by induction of apoptosis after prolonging the incubation to 24 h. The prospects of using HepaRG cells cultured in spheroids for estimation of drug toxicity by variations in the expression of stress-dependent genes were demonstrated. An increase in expression of genes of GSR and HSPA1A proteins at the early stages of incubation with cisplatin can serve as a marker of the cytotoxic effects of cisplatin and other agents with similar mechanisms of action.